1. Identification in human osteoarthritic chondrocytes of proteins binding to the novel regulatory site AGRE in the human matrix metalloprotease 13 proximal promoter.
- Author
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Fan Z, Tardif G, Boileau C, Bidwell JP, Geng C, Hum D, Watson A, Pelletier JP, Lavigne M, and Martel-Pelletier J
- Subjects
- Base Sequence, Cartilage, Articular chemistry, Cartilage, Articular pathology, Cells, Cultured, Chondrocytes pathology, Collagenases analysis, DNA-Binding Proteins analysis, Fibroblasts enzymology, Fibroblasts pathology, Humans, Matrix Metalloproteinase 13, Molecular Sequence Data, Osteoarthritis, Knee pathology, Osteoarthritis, Knee surgery, Synovial Membrane chemistry, Synovial Membrane enzymology, Synovial Membrane pathology, Trans-Activators analysis, Trans-Activators metabolism, Cartilage, Articular enzymology, Chondrocytes enzymology, Collagenases metabolism, DNA-Binding Proteins metabolism, Osteoarthritis, Knee metabolism, Promoter Regions, Genetic physiology
- Abstract
Objective: Matrix metalloprotease 13 (MMP-13) plays a major role in osteoarthritic (OA) processes. We previously identified the AG-rich element (AGRE) regulatory site (GAAAAGAAAAAG) in the proximal promoter of this gene. Electrophoretic mobility shift assays (EMSAs) done with nuclear extracts from OA chondrocytes showed the presence of 2 AGRE protein-binding complexes, the formation of which depended on the pathophysiologic state (high or low) of the cells; the low OA (L-OA) chondrocytes have low MMP-13 basal levels and high interleukin-1beta (IL-1beta) inducibility, and the high OA (H-OA) chondrocytes have high MMP-13 basal levels and low IL-1beta inducibility. In this study, we sought to determine the importance of individual AGRE bases in promoter activity and to identify AGRE binding proteins from L-OA and H-OA chondrocyte complexes., Methods: Promoter activity was determined following transient transfection into human OA chondrocytes. AGRE binding proteins were identified by mass spectroscopy., Results: Individual mutations of the AGRE site differentially modulated promoter activity, indicating that the intact AGRE site is required for optimal MMP-13 expression. Damage-specific DNA binding protein 1 (DDB-1) was identified in the L-OA chondrocyte-binding complex. EMSA experiments performed with the mutation of the left AGRE site (GTGCTGAAAAAG) and nuclear extracts of L-OA chondrocytes reproduced the pattern seen in the H-OA chondrocytes. Mass spectroscopy identified p130cas as one of the proteins in this complex. Supershift experiments showed the presence of p130cas and nuclear matrix transcription factor 4 (NMP-4) in the wild-type AGRE/H-OA chondrocyte complex., Conclusion: These data suggest that the binding of p130(cas) and NMP-4 to the AGRE site regulates MMP-13 expression and may trigger the change in human chondrocytes from the L-OA state to the H-OA state.
- Published
- 2006
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